Optimisation of cell and ex vivo culture conditions to study vascular calcification

Abstract

Medial vascular calcification (MVC) is a highly prevalent disease associated with a high risk of severe, potentially lethal, complications. While animal studies may not systematically be circumvented, in vitro systems have been proven useful to study disease physiopathology. In the context of MVC, the absence of a clinically relevant standardized in vitro method prevents the appropriate comparison and overall interpretation of results originating from different experiments. The aim of our study is to establish in vitro models mimicking in vivo vascular calcification and to select the best methods to unravel the mechanisms involved in MVC. Human aortic smooth muscle cells and rat aortic rings were cultured in different conditions. The influence of fetal calf serum (FCS), alkaline phosphatase, phosphate and calcium concentrations in the medium were evaluated. We identified culture conditions, including the herein reported Aorta Calcifying Medium (ACM), which allowed a reproducible and specific medial calcification of aortic explants. Studying cells and aortic explants cultured, the involvement of bone morphogenetic protein 2 (BMP2) pathway, fibrosis and apoptosis processes in in vitro MVC were demonstrated. Expression of osteoblastic markers was also observed suggesting the occurrence of transdifferentiation of smooth muscle cells to osteoblasts in our models. The use of these models will help researchers in the field of vascular calcification to achieve reproducible results and allow result comparison in a more consistent way.

Fig 1. Influence of Fetal Calf Serum (FCS) and phosphate concentrations on calcium deposition in HASMC.

Von Kossa staining after 14 days of culture with or without cells. No calcium staining was observed in low phosphate containing media. Calcium staining was observed in high P media, in the absence of FCS both, in the presence as well as in the absence of cells depicting a passive mechanism (no need for cells to obtain phosphocalcic deposition). In high phosphate concentration (3.8 mM) and FCS supplemented wells, no calcium was observed in the absence of cells whilst a restricted positive calcium staining was observed in the presence of cells, suggesting the involvement of an active process (cell related) in the appearance of the calcium deposits, expected to be more representative of MCV disease. (Original magnification x200).

Fig 2. Influence of cell culture medium composition on calcification of HASMC.

A) Influence of the phosphate donor in medium supplemented with 15%FCS. No calcium was observed in the absence of cells (wells of the lower panel). While significant amounts of Ca were observed in the cell cultures (upper panel). The higher calcium deposit on cells was observed with basic (Na₂HPO₄), compared to acidic (NaH₂PO₄) or neutral pH (NaH₂PO₄/ Na₂HPO₄) phosphate buffers (* P<0.01 versus others; ** P<0.001 versus others). B) Influence of calcium concentration in medium supplemented with 15%FCS and high phosphate (3.8 mM). Calcium deposition was proportional to the Ca content in the medium in the media with 3.8 mM phosphate (* P<0.01 versus others; ** P<0.001 versus others). C) Influence of phosphate donor, phosphate level and calcium level in medium supplemented with 15%FCS. High phosphate concentration (3.8 mM) alone induced a significant calcium deposition in cell culture, while high Ca (2.4 mM) alone did not. High phosphate associated with high Ca medium resulted in the highest calcium deposition; the positive Ca staining in the absence of cells (wells in the lower panels) suggests also the participation of a passive phosphocalcic deposition. Calcium deposition was again higher with basic phosphate donor (Na₂HPO₄) buffer compared to the other phosphate donors (* P<0.001 versus other P/Ca conditions with the same P donor; ¤ P<0.001 versus other P donors with the same P/Ca condition). D) Influence of Alkaline phosphatase (ALK) and FCS in high phosphate and high Ca medium. ALK increased calcium deposition in the presence of FCS, both in the presence of cells as well in the absence of cells (¤ P<0.05 for the effect of cells compared to the same FCS and ALK conditions; # P<0.001 for the effect of FCS compared to the same cell and ALK conditions; * P<0.05 for the effect of ALK compared to the same cell and FCS conditions).

Fig 3. Pro-osteogenic pathway exploration in HASMC cultivated in calcifying or control medium.

A) BMP2 and BMPR1A RNA expression in HASMCs by qPCR after 48 hours of culture. Both gene expressions were increased in cells cultured in calcifying medium (high phosphate, high calcium, high FCS) compared to the control medium. Gene expression was normalized to GAPDH expression. B) Exploring BMP2 signalling pathway by Western blot of phospho-Smad1 protein. The expression of phospho-Smad1 was increased in calcifying medium compared to control medium (* P<0.01 at day 3 and day 7). C) Relative quantification of gene expression by qRT-PCR analyses. The expression of runt-related transcription factor 2 (RUNX2), receptor activator of nuclear factor kappa-B ligand (RANKL), osteopontin (OPN), osteoprotegerin (OPG) and alkaline phosphatase (ALP) genes was increased in calcifying medium compared to control medium. (Gene expression was normalized to GAPDH expression; CTL: control medium; CM: calcifying medium).

Fig 4. Influence of Fetal Calf Serum (FCS) and phosphate concentrations on calcium deposition in aortic rings.

Von Kossa staining of aortic rings after 14 days of culture in the absence or presence of 15% FCS, both in basal medium or high phosphate (3.8 mM) medium. Note a clear von Kossa positive band following the distribution of the medial layer of the aortic ring only in the presence of FCS and high phosphate medium. (Original magnification x40).

Fig 5. Influence of medium composition on ex vivo calcification in aortic rings culture.

A) Influence of the phosphate donor (basic, neutral or acidic). Calcium incorporated into the tunica media of aortic rings assessed by von Kossa staining was higher with neutral phosphate donor buffer (NaH₂PO₄ / Na₂HPO₄), P = 0.08. B) Effect of calcium and phosphate concentrations on calcification. A marked increase in calcium content was observed in high phosphate/high calcium conditions, whilst no increase in von Kossa staining positive surface area was observed when compared with high phosphate, suggesting that a significant proportion of calcium was not incorporated into the tunica media (* P<0.01 versus others; ** P<0.001 versus others). C) Effect of alkaline phosphatase on calcification. In 15% FCS supplemented cultures, calcium deposition decreased in the presence of alkaline phosphatase (# P<0.001 for the effect of FCS compared to the same ALK condition; * P<0.01 for the effect of ALK compared to the same FCS condition). (original magnifications x40).

Fig 6. Mechanisms participating in calcium deposition.

A) Calcium deposits, fibrosis and apoptosis: Aortic rings cultured in calcifying medium were positively stained with von Kossa, Sirius red and TUNEL staining, suggesting that fibrosis and apoptosis occur in calcium deposits induced ex-vivo in aortic rings in culture. B) Pro-osteogenic pathway exploration in ex-vivo aortic ring calcification in culture. Phospho-Smad1, Osteopontin and Osteocalcin expressions were observed in aortic rings cultured in calcifying medium (ACM), whilst were not observed in control medium (CTL) suggesting that calcification process involves BMP2 pathway and trans-differentiation to osteoblastic phenotype (original magnifications x40).